Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Involving particle-mediated transfecion
Reexamination Certificate
1998-09-23
2002-11-26
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Involving particle-mediated transfecion
C800S320000, C800S320200, C435S470000, C435S419000, C435S430000
Reexamination Certificate
active
06486384
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to compositions and methods for the in vitro culture, transformation, and regeneration of plants.
Genetic improvement of various crop species by genetic engineering has sometimes been hindered because techniques for in vitro culture, transformation, and regeneration of amenable cultivars are less effective with recalcitrant commercial cultivars.
The ability to genetically engineer monocots, including cereal crops, to improve their performance and pest-resistance or to enhance alternative uses is of great importance. The practical utility of stable transformation technologies is largely dependent on the availability of efficient methods for generating large numbers of fertile green plants from tissue culture materials.
Virtually all current genetic engineering technologies require that genes be delivered to cells grown in vitro. Most published methods for generating fertile transformed plants from cereals (e.g. rice, wheat, maize, oat, sorghum, triticale, barley and rye) utilize as initial explants the immature scutellum of the embryo or microspores directly or tissue derived from immature embryos or microspores. From these initial explants, cellular proliferation occurs. After selection or screening for transformants, plants are regenerated.
Five critical problems adversely impact the utility of these transformation methods, particularly monocot species such as cereals and grasses. The first is heritable variability, termed “somaclonal variation” which results from spontaneous and heritable genetic changes in cultured plant tissues. Somaclonal variation can adversely affect the field performance (e.g., height, yield, and seed weight) of tissue culture-derived plants. Somaclonal variation can also limit the use of transgenic plants for breeding, since introgression of transgenes from such plants into acceptable genetic backgrounds can require multiple cycles of hybridization and progeny analysis, particularly if one or more heritable mutations were closely linked to the transgene or to genes controlling other critical traits.
The second problem is related to the increase in the incidence of albino plants caused by, for example, the physiological and biochemical changes imposed by selection, by the increased time frame required for selection during transformation and/or with changes in genotype. With some genotypes published methods of transformation result in the selection of transformed callus that is either nonregenerable or regenerates albino plants.
The third problem is the genotype dependence of the in vitro culture response of various explants, e.g. scutellum- and microspore-derived tissues and the resulting difficulties in applying transformation methods developed for amenable genotypes to commercially important more recalcitrant genotypes.
The fourth problem is related to the instability of the introduced genes themselves or of the expression of the transgenes. In transgenic cereals the introduced gene is sometimes lost in subsequent generations and there can also be a loss of the ability of the plant to express a transgene.
The fifth problem is that transgenic plants produced by published methods are often polyploid and therefore cannot be propagated as the more desirable diploid varieties, further limiting the usefulness of transgenic plants in breeding programs.
Aspects of the in vitro culturing and/or transformation process are likely to be responsible for or related to these and other problems encountered in efforts to genetically engineer plant species, including monocots such as cereals and grasses. Most transformation protocols require that the target tissue undergo embryogenesis, which may include de-differentiation of a single original transformed cell before the sustained cell divisions that give rise to an embryo consisting mostly or entirely of cells that contain the introduced DNA. De-differentiation during in vitro culturing introduces stresses on the genome, causing modifications of the genome that are associated with somaclonal variation, including DNA methylation, point mutations, deletions, insertions, and the generation of gross cytogenetic abnormalities. These genomic modifications lead to subsequent phenotypic abnormalities and performance losses and may contribute to the other problems listed above.
Transformation methods using excised shoot apices have been previously described (see, for example, U.S. Pat. No. 5,164,310 to Smith et al.; Zhong et al., 1996). However, these methods have not proven to be effective for some monocots, including commercially important varieties of barley, oat and wheat. For example, until now there is no method known for transformation of the spring barley cultivar (
Hordeum vulgare
L.) Harrington, which is a widely grown two-rowed malting barley.
There is a need, therefore, for improved methods for plant transformation and regeneration, particularly for use with monocot species such as cereals and grasses.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for plant transformation and regeneration that are applicable to a wide variety of monocots, including commercially important cereal genotypes that have proven difficult or impossible to transform and regenerate by previously available methods. These improved methods result in significantly higher regeneration frequencies, reduced somaclonal variation, improved transgene expression stability, and reduced albinism.
The invention is based on the discovery that transgenic plants produced by transformation of organogenic tissues, particularly tissues derived from shoot meristematic cells, are generally healthier than plants produced by conventional transformation of embryogenic tissues. In particular, transgenic plants produced by transformation methods provided by the invention may be more stable, exhibit fewer problems associated with methylation, show fewer mutations caused by somaclonal variation, and exhibit reduced albinism. In addition, the transformation methods disclosed are applicable to commercial varieties of monocots such as barley, wheat and oat that are recalcitrant to conventional transformation methods.
The transformation method disclosed relies on introducing the nucleic acid sequence (generally referred to as the “transgene”) into shoot meristematic tissue that is typically derived from a shoot apex or a leaf base. This tissue requires little or no de-differentiation in order to regenerate plants that express the transgene. Thus, in contrast to embryogenic callus tissue (a conventional target for transformation), these meristematic tissues do not undergo significant de-differentiation in the transformation process. Rather, these cells require only a simple redirection of growth in order to produce whole transgenic plants. The present invention also provides plant growth media containing growth substrates (including suitable levels of plant hormones and other components) with which the efficient production and regeneration of this meristematic tissue can be achieved. In particular, the invention provides media suitable for the production of meristematic tissue that is highly amenable to transformation from cultivars of monocots that are otherwise recalcitrant to transformation.
In general terms, the transformation method provided by the invention comprises obtaining shoot meristematic tissue from the plant to be transformed. Any source of shoot meristematic tissue may be employed, including shoot meristematic tissue taken from shoot apices of embryos and seedlings or axillary or adventitious shoots.
The meristematic tissue is incubated in the light on a meristem proliferation medium (MPM) to induce production of adventitious meristematic cells, which are then used as the target for nucleic acid transformation. Transformation may be achieved by any effective means, including for example conventional particle bombardment. MPM promotes fast growth of meristematic cells without promoting shoot or root formation. Particular compositions of MPM that are provided by this invention incl
Bregitzer Phillip
Cho Myeong-Je
Lemaux Peggy G.
Zhang Shibo
Fox David T.
Kruse David H
Morrison & Foerster / LLP
The Regents of the University of California
LandOfFree
Methods and compositions for transformation of cereals using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and compositions for transformation of cereals using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and compositions for transformation of cereals using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2939775